The present invention relates to an immunoassay for the brain natriuretic peptide (BNP) which is a member of natriuretic peptide family, more specifically, it relates to an immunoassay for xcex3-BNP and derivatives thereof.
Natriuretic peptide family includes three members, i.e., atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and type C natriuretic peptide (CNP). Among them, ANP and BNP are cardiac hormones which are mainly biosynthesized in and secreted from the heart. ANP and BNP are similar in structure. ANP is a peptide of 28 amino acids with a ring (circular) structure formed by a disulfide bond between the 7th and the 23rd cysteine residues, while BNP is a peptide of 32 amino acids with a ring structure formed by a disulfide bond between the 10th and the 26th cysteine residues. These mature peptides of 28 and 32 amino acids have been considered to be produced from respective precursor when a leader sequence is cleaved off intracellularly or at the time of secretion. That is, there has been reported that human BNP is first synthesized as a preprohormone (hereinafter, referred to as prepro-BNP) in myocardial cells, which is split before or at the time of secretion between Ser26-His27 to give pro-BNP (hereinafter, referred to as xcex3-BNP), and which is further split between Arg102-Ser103 to give BNP-32 (hereinafter, referred to as xcex1-BNP) and BNP(1-76), and that the former exhibits the activity. It has been considered that at least the ring structure must be remained for the expression of activity.
The secretion of cardiac hormones being stimulated by various heart diseases, it well reflects the change in the cardiac functions. The secretion of ANP is accelerated mainly when the atrium undergoes a load, while the biosynthesis and secretion of BNP are stimulated when the ventricle undergoes a load. Accordingly, both ANP and BNP are useful as indicators in the diagnosis of heart disease. As the progress of investigation in the in vivo role of respective hormone, the advantageous features of BNP as an indicator for diagnosing heart disease have become clear. For example, the blood concentration of BNP is only ⅙ of ANP in a normal subject but it becomes higher than ANP in patients of heart failure or the like; the blood concentration of BNP increases in the case of heart failure like ANP, and the plasma concentration of BNP often exceeds that of ANP reflecting more accurately the severity of heart dysfunction; the plasma concentration of both ANP and BNP elevates in peripheral blood and elevation rate is higher in BNP. Moreover, BNP level in patients of heart failure sometimes increases to several tens times to several hundreds times of that of healthy normal subjects, and the change of BNP in the cases of heart failure is so marked that no other hormones are incomparable therewith. For these reasons, the usefulness of BNP measurement has been suggested (Y. Saito et al., Mebio, 12(5), 28, 1995).
Under the conditions, an immunoassay which utilizes anti-BNP antibody and is applicable to the diagnosis of cardiac insufficiency has been proposed. Japanese Patent Publication (KOHYO) 7-507210 describes a method of measuring xcex3-BNP (1-76) produced by biodegradation by protease or the like. However, this method is directed to xcex3-BNP (1-76) which lacks the portion(s) essential for the expression of activity such as ring structure and, therefore, cannot determine the hormone activity directly.
An assay kit for the measurement of xcex1-BNP having natriuretic activity has been marketed (xe2x80x9cBNP-32xe2x80x9d, Peninsula). With this kit, degradation products of xcex1-BNP in blood including fragments lacking activity due to the deletion of C-terminal region can also been measured. Taking the low blood concentration of BNP into consideration, the measurements involving the degradation products cannot be disregard. Accordingly, the said method connotes disadvantages to be an assay for BNP in th establishment of an accurate diagnosis of heart failure.
As a kit for the measurement of BNP free from the disadvantages above has been marketed (xe2x80x9cSHIONORIA BNPxe2x80x9d, Shionogi), which characteristically uses an antibody recognizing the structure essential for the expression of activity. However, this method would be affected significantly by the process for collecting and storing blood sample, because xcex1-BNP is extremely instable in collected blood. It is, therefore, suggested that the sample should be specifically treated by, for instance, adding an agent for inhibiting degradation into a blood collecting tube or maintaining the sample at low temperature so as to obtain reliable data. Such procedures may hamper the extensive clinical application of the said BNP assay kit.
The present inventors have conducted research intensively for the purpose of establishing an accurate method of diagnosing cardiac diseases involving BNP and found that BNP exists in blood in the form of xcex3-BNP or its degradation product which at least comprises structurally the xcex1-BNP moiety (hereinafter, they are referred to as xe2x80x9cxcex3-BNP derivativexe2x80x9d), and not in the form of xcex1-BNP which has so far been considered to be dominant. The inventors have also found that xcex3-BNP is more stable than xcex1-BNP in blood, that is, one role of the N-terminal structure of xcex3-BNP, among many, would be the stabilization of BNP. The above indicates that an organism biosynthesizes at least 2 kinds of BNP molecule which share the BNP activity but differ in half-life. These findings led the present inventors to have a view that it is indispensable to establish a method specific for not only xcex1-BNP but also xcex3-BNP to accomplish an accurate diagnosis of cardiac diseases.
The present invention provides an immunoassay specific for mammalian xcex3-BNP derivatives, characterized in that it uses the first antibody which is reactive with mammalian xcex1-BNP and the second antibody which is reactive with mammalian prepro-BNP or xcex3-BNP derivatives and not reactive with xcex1-BNP.
As used herein, the term xe2x80x9cmammalian xcex1-BNPxe2x80x9d refers to a peptide of low molecular weight having natriuretic activity which is derived from mammalian prepro-BNP or xcex3-BNP through the removal of N-terminal region as a result of processing at the carboxy terminus of processing signal sequence. In case of human BNP, xcex1-BNP is a peptide consisting of C-terminal 32 amino acids (Nos. 103-134) of the amino acid sequence of SEQ ID NO: 1 and having a ring structure. The carboxy terminus of processing signal sequence on the prepro-BNP molecule varies slightly depending on the species. For example, it is No. 102 Arg in case of human BNP while it is No. 100 amino acid in case of porcine or canine BNP.
As used herein, the term xe2x80x9cmammalian xcex3-BNPxe2x80x9d refers to a pro-BNP comprising a partial peptide of 32 amino acids corresponding to xcex1-BNP at the carboxy terminal region. In case of human xcex3-BNP, it is pro-BNP of 108 amino acids from No. 27 His to No. 134 His of the amino acid sequence of SEQ ID NO: 1. The term xe2x80x9cprepro-BNPxe2x80x9d refers to a peptide of 134 amino acids from No. 1 Met to No. 134 His of the amino acid sequence of SEQ ID NO: 1 in case of human.
As used herein, the term xe2x80x9cmammalian xcex3-BNP derivativexe2x80x9d refers to a peptide fragment derived from mammalian prepro-BNP or xcex3-BNP through mainly the in vivo protease reaction, which fragment includes or is larger than xcex1-BNP. Although xcex3-BNP derivative would comprise a molecule of the same or smaller size compared to xcex3-BNP in general, it may comprise a molecule larger than xcex3-BNP. Otherwise specifically mentioned, as used herein, the term xe2x80x9cxcex3-BNP derivativexe2x80x9d includes xcex3-BNP itself.
The term xe2x80x9cstablexe2x80x9d, when used herein in connection with BNP, means that a BNP molecule maintains the C-terminal ring structure including C-terminus of BNP and the natriuretic activity after undergoing the degradation by protease, and that the said activity is not significantly decreased even 24 hours from the collection of blood samples. In light of this definition, the xcex3-BNP derivative as the target substance (analyte) of the present immunoassay is stable.
On the other hand, the term xe2x80x9cunstablexe2x80x9d means that a BNP sample undergoes degeneration by protease at the C-terminal region and that the natriuretic activity is significantly decreases 24 hours from the collection of blood samples. In light of this definition, xcex1-BNP is unstable.